Homeobox genes encode transcription factors that control tissue patterning and morphogenesis, yet their role in pulmonary vascular development remains obscure. Our preliminary studies indicate that the paired related homeobox gene, Prxl, is required for lung vascularization via its ability to promote both endothelial cell (EC) differentiation and vascular network formation. The Specific Aims of this proposal are as follows: (1) To define how Prxl drives EC differentiation during lung vasculogenesis: Tie-GFP transgenic mice will be used to locate Prxl-expressing ECs throughout lung development, and chromatin immunoprecipitation assays will be used to identify direct targets for Prxl in differentiating lung ECs. The validity and functions of Prxl and its targets will be evaluated using Prxl-null mice and tissue culture models of pulmonary EC differentiation; (2) To determine how Prxl-dependent induction of the extracellular matrix protein tenascin- C (TN-C) promotes lung vascular network formation: Knockout studies using fetal lung explants, yeast 2- hybrid assays and tissue recombinations will be used to understand how TN-C promotes Prxl-dependent network formation; (3) To delineate how focal adhesion kinase (FAK) controls Prxl during vascular network formation: Adenoviral-based inhibition of FAK activity, and dissection of the Prxl gene promoter will be used to comprehend how FAK controls Prxl transcription and vascular morphogenesis in the lung. Overall, this proposal will result in a detailed understanding of the role of Prxl throughout fetal lung vascularization. This study should provide new concepts in lung vascular biology, and will hopefully result in novel diagnostic tools and therapies for the treatment of newborn and adult diseases in which the pulmonary vasculature is compromised, eg. bronchopulmonary dysplasia and pulmonary hypertension.